γδT细胞对肺巨噬细胞的免疫调节作用的研究

发布时间：2018-06-15 17:05

本文选题：脾脏γδT细胞 + 抗原　；参考：《北京协和医学院》2011年硕士论文

【摘要】：T淋巴细胞表面表达两种抗原受体(TCR)即αβTCR和γδTCR。人们目前研究较多的是αβT细胞,γδT细胞所占比例较小,仅占正常人外周血淋巴细胞总数的3%～10%,但是在机体免疫调节及免疫监视中起着重要的作用。γδT细胞主要识别以下类型的抗原：MHC分子；磷酸化抗原；热休克蛋白；非肽类抗原等。本文首先利用三种不同的抗原(即IPP,HSP70和EP6)刺激小鼠脾脏γδT细胞,以了解三种抗原在不同浓度和不同方式干预下小鼠脾脏γδT细胞的增殖纯度。实验中主要采用流式细胞术检测细胞培养中脾脏γδT细胞的比例。结果表明,在IPP,HSP70和EP6持续刺激下脾脏γδT细胞在培养第三天时均出现增殖的高峰,而后增殖有所下降。随着三种抗原浓度的增加,IPP的浓度在1.25ng/ml时出现脾脏γδT细胞的增殖高峰,扩殖的百分比为5.45%±0.02%(P0.05)；而后随着浓度的增加增殖减低。在相同浓度下不同干预方式中发现,三种抗原一次性单次刺激时亦均在作用后第三天出现增殖高峰,扩增的百分比分别为3.60%±0.27%,4.54%±0.16%,3.20%±0.11%(P0.05)；IPP和EP6组第三天增殖高峰时,持续性刺激的增殖纯度大于一次性单次刺激,扩增的百分比分别为(6.38%±0.58%VS 3.60%±0.27%,5.41%±0.11% VS 3.20%±0.11%)(P0.05)。本实验证实了在体外刺激脾脏γδT细胞时,增殖高峰出现在培养的第三天。不同抗原刺激的γδT细胞种类不同,因而增殖的纯度亦不相同。持续性的抗原刺激较一次性单次刺激更能促进γδT细胞的增殖。 γδT细胞的免疫调节作用越来越受研究者的重视,已知另一种在免疫反应中起重要作用的巨噬细胞广泛分布于全身各组织中,是机体抵抗外界入侵的重要防线。雷帕霉素是一种大环内酯类免疫抑制剂,已知的主要靶点是胞浆内的激酶mTOR。通过体外培养LPS诱导的小鼠急性肺损伤模型中脾脏γδT细胞和肺组织巨噬细胞,以了解脾脏γδT细胞和肺组织巨噬细胞之间是否存在相互作用以及体外雷帕霉素对这种相互作用是否有影响。实验中,建立LPS诱导的小鼠急性肺损伤的模型,采用HE染色固定肺组织并观察其病理改变；流式细胞术检测磁珠分选前后肺组织巨噬细胞和脾脏γδT细胞的纯度,荧光显微镜下观察脾脏γδT细胞的形态,ELISA法检测小鼠脾脏γδT细胞和肺组织巨噬细胞培养的上清液中炎症因子IL-4,IL-12,IFN-γ和TNF-α的分泌水平；Real-time PCR检测培养细胞中IL-4,IL-12,IFN-γ和TNF-α的mRNA水平。实验结果显示在LPS诱导的急性肺损伤模型中,LPS组支气管肺泡灌洗液的总细胞和中性粒细胞浓度明显大于PBS组和LPS+RAPA组,总细胞分别为(85.6±31.2VS 10.8±2.5,48.6±26.0)×104个/ml,中性粒细胞的分别为(73.4±28.8 VS 1.5±0.5,39.8±24.8)×104个/ml(PO.05)。脾脏γδT细胞与肺组织巨噬细胞按1:1混合培养时,LPS组上清液IFN-γ高于PBS组,RAPA组和LPS+RAPA组(174.17±42.16 VS 16.80±8.80,12.19±4.14,7.16±3.03)pg/ml,(P0.05)。IL-4高于PBS组和LPS+RAPA组(73.82±61.71 VS 19.19±10.01,12.35±1.86)pg/ml,(P0.05)。而TNF-α只高于RAPA组和LPS+RAPA组(34.24±8.08 VS 12.20±4.56,10.56±2.99)pg/ml,(P0.05)。混合培养时LPS组IFN-γmRNA的表达明显高于PBS组(10.67±4.62 VS 1),(P0.05)。本研究中体外将脾脏γδT细胞和肺组织巨噬细胞分离纯化后进行培养,使得细胞生长的内环境受外界干扰较少,较真实的反映细胞的生物学活性。本实验结果证实了体内实验中RAPA能抑制LPS诱导小鼠急性肺损伤的支气管肺泡灌洗液中总细胞和中性粒细胞的增殖,体外实验中显示了脾脏γδT细胞和肺组织巨噬细胞单独培养时未见明显的变化,但混合培养时细胞因子分泌产生很大变化,说明脾脏γδT细胞和肺组织巨噬细胞可能存在细胞间的相互作用从而影响了各自的生物学活性。在混合培养时,LPS能刺激脾脏γδT细胞和肺组织巨噬细胞分泌较多的IFN-γ和IL-4,并能增加混合培养细胞中IFN-γmRNA的表达。RAPA能抑制混合培养分泌的IFN-γ,IL-4和TNF-α,说明RAPA抑制了脾脏γδT细胞和肺组织巨噬细胞的相互作用,该抑制作用对基因转录水平没有任何影响。其具体作用机制还需要我们在今后的研究中探讨以寻找炎症疾病治疗的新靶点。
[Abstract]:On the surface of T lymphocyte, two kinds of antigen receptor (TCR), that is, alpha beta TCR and gamma delta TCR., are widely studied. The proportion of gamma delta T cells is small, which accounts for only 3% to 10% of the total number of lymphocytes in normal human peripheral blood, but plays an important role in immune regulation and immune surveillance. The antigen: MHC molecule, phosphorylation antigen, heat shock protein, non peptide antigen and so on. First of all, three different antigens (IPP, HSP70 and EP6) were used to stimulate the spleen Delta T cells of the spleen in order to understand the proliferation purity of the three kinds of antigen in the spleen of the spleen of the mice under the intervention of different concentrations and different ways.
In the experiment, the proportion of splenic Delta T cells in cell culture was detected by flow cytometry. The results showed that the proliferation peak of splenic Delta T cells in IPP, HSP70 and EP6 stimulated third days, and then the proliferation decreased. With the increase of the concentration of three antigens, the concentration of IPP in 1.25ng/ml appeared in the spleen of the spleen Delta T fine. The proliferating peak of the cell was 5.45% + 0.02% (P0.05), and then the proliferation decreased with the increase of concentration. At the same concentration, the proliferation peak was found at third days after the action of three kinds of antigen, 3.60% + 0.27%, 4.54% + 0.16%, 3.20% + 0.11% (P0. 05); when the proliferation peak at third days in IPP and EP6 groups, the purity of the sustained stimulation was greater than that of one time single stimulus, and the percentage of the amplification was (6.38% + 0.58%VS 3.60% + 0.27%, 5.41% + 0.11% VS 3.20% + 0.11%) (P0.05).
The experiment confirmed that the proliferation peak appeared at third days in vitro stimulation of the spleen Delta T cells. The species of gamma delta T cells stimulated by different antigens were different, and the purity of the proliferation was different.
The immunomodulatory effect of gamma delta T cells is becoming more and more important. Another known macrophage, which plays an important role in the immune response, is widely distributed throughout the body and is an important defense against the invasion of the outside world. Rapamycin is a macrolide immunosuppressant. The main target is the kinase mT in the cytoplasm. OR. through in vitro culture of LPS induced mouse acute lung injury model of splenic Delta T cells and lung tissue macrophages, in order to understand whether there is a interaction between the splenic Delta T cells and the macrophages of the lung tissue and whether there is any effect of rapamycin on this interaction in vitro.
In the experiment, the model of LPS induced acute lung injury in mice was established. The lung tissue was fixed by HE staining and its pathological changes were observed. The purity of the macrophages and splenic Delta T cells in the lung tissue were detected by flow cytometry before and after the separation of magnetic beads. The morphology of the splenic Delta T cells was observed under the fluorescence microscope. The spleen Delta T cells and lungs of the mice were detected by ELISA method. The secretion level of inflammatory factors IL-4, IL-12, IFN- gamma and TNF- alpha in supernatant of macrophage culture was organized, and Real-time PCR was used to detect mRNA levels of IL-4, IL-12, IFN- gamma and TNF- alpha in cultured cells.
The results showed that in the model of LPS induced acute lung injury, the concentration of total cells and neutrophils in the bronchoalveolar lavage fluid in group LPS was significantly greater than that of group PBS and LPS+RAPA, and the total cells were (85.6 + 31.2VS 10.8 + 2.5,48.6 + 26) x 104 /ml respectively, and the neutrophils were divided into (73.4 + 28.8 VS 1.5, 0.5,39.8 + 24.8) x 104 /ml (PO.) 05) when the splenic Delta T cells and macrophages were mixed with 1:1 in the lung tissue, the supernatant of the LPS group was higher than the PBS group, the RAPA group and the LPS+RAPA group (174.17 + 42.16 VS 16.80 + 8.80,12.19 + 4.14,7.16 + 3.03) pg/ml, which were higher than those of 73.82 + 61.71 (73.82 + 61.71 and 19.19 + 1.86). Group and LPS+RAPA group (34.24 + 8.08 VS 12.20 + 4.56,10.56 + 2.99) pg/ml, (P0.05). The expression of IFN- y mRNA in group LPS was significantly higher than that in group PBS (10.67 + 4.62 VS 1) at mixed culture, (P0.05).
In this study, the splenic Delta T cells and lung tissue macrophages were isolated and purified in this study. The internal environment of the cell growth was less disturbed and the biological activity of the cells was truly reflected. The results of the experiment confirmed that in the experiment, RAPA could inhibit the bronchoalveolar lavage fluid induced by LPS in the acute lung injury of rats. The proliferation of total and neutrophils showed that there was no obvious change in the splenic Delta T cells and lung tissue macrophages in vitro, but the secretion of cytokines produced a great change in mixed culture, indicating that there might be intercellular interactions between the splenic Delta T cells and the macrophages in the lung tissue. In mixed culture, LPS stimulates the secretion of more IFN- gamma and IL-4 by macrophages in the spleen Delta T cells and lung tissue, and can increase the expression of IFN- gamma mRNA in mixed culture cells, which can inhibit the IFN- gamma, IL-4 and TNF- alpha in mixed culture, indicating that RAPA inhibits the interaction between the spleen and the macrophages of the lung tissue. The inhibitory effect has no effect on the transcriptional level of the gene. Its specific mechanism also requires us to explore new targets for the treatment of inflammatory diseases in future studies.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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